RESEARCH ARTICLE
Withania somnifera Root Extract Enhances Chemotherapy through ‘Priming’ Aine Brigette Henley1¤, Ling Yang2, Kun-Lin Chuang2, Meliz Sahuri-Arisoylu1, LiHong Wu3, S. W. Annie Bligh1*, Jimmy David Bell1 1 Department of Life Sciences, Faculty of Science and Technology, University of Westminster, London, United Kingdom, 2 Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung, Taiwan, 3 Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China ¤ Current address: Department of Microbiology, Tumour and Cell Biology, Karolinska Institutet, Stockholm, Sweden * [email protected]
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OPEN ACCESS Citation: Henley AB, Yang L, Chuang K-L, SahuriArisoylu M, Wu L-H, Bligh SWA, et al. (2017) Withania somnifera Root Extract Enhances Chemotherapy through ‘Priming’. PLoS ONE 12(1): e0170917. doi:10.1371/journal.pone.0170917
Abstract Withania somnifera extracts are known for their anti-cancerous, anti-inflammatory and antioxidative properties. One of their mechanisms of actions is to modulate mitochondrial function through increasing oxidative stress. Recently ‘priming’ has been suggested as a potential mechanism for enhancing cancer cell death. In this study we demonstrate that ‘priming’, in HT-29 colon cells, with W. somnifera root extract increased the potency of the chemotherapeutic agent cisplatin. We have also showed the W. somnifera root extract enhanced mitochondrial dysfunction and that the underlying mechanism of ‘priming’ was selectively through increased ROS. Moreover, we showed that this effect was not seen in non-cancerous cells.
Editor: Irina V Lebedeva, Columbia University, UNITED STATES Received: August 22, 2016
Introduction
Accepted: January 12, 2017
Cancer is one of the major causes of death around the globe, despite the progress observed in surgery, radiation and chemotherapy [1–3]. Cancer progression relies on the ability of cancer cells to exploit the normal physiological processes of the host [1]. This unfortunately means that the cytotoxicity of chemotherapeutic drugs is not limited to cancer cells and can affect non-cancer cells [1, 4]. Today the most common therapeutic strategy of chemotherapy drugs is the use of two drugs in combination, however this is invariably associated with side effects which include chemoresistance [5–7]. It is therefore essential to explore and enhance current methods of chemotherapy to improve their efficacy while also reducing side effects. One such approach is ‘priming’, whereby cancer cells are pre-treated with a ‘priming’ agent (curcumin, quercetin, aspirin) prior to chemotherapy treatment [8–12]. The underlying mechanism underpinning ‘priming’ appears to be the enhancement of cell death through mitochondrial dysfunction [8, 9]. Mitochondrial dysfunction can alter ROS levels, ATP production and overall cell viability and is a novel key target in cancer treatment [9, 13, 14]. Withania somnifera is an Ayurvedic medicinal plant whose root and leaves extracts have been used for its antioxidant and restorative properties as well as to reduce cancer growth
Published: January 27, 2017 Copyright: © 2017 Henley et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors received no specific funding for this work. Competing Interests: The authors have declared that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0170917 January 27, 2017
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’Priming’ with Withania somnifera
[2, 6, 15]. W. somnifera extracts have been found to be effective in treating several types of cancer including skin, leukaemia, breast, colon and pancreas [15–21]. However, the mechanisms of action have yet to be fully elucidated, but indications of involvement in mitochondrial membrane permeability have been reported in several studies [1, 21–23]. Additionally, W. somnifera extracts have been shown to increase reactive oxygen species (ROS) [1, 19, 23]. The mitochondria is an important regulator of cell survival and progression and is the main source of ROS which is linked with mitochondrial function [24]. Cancer cells metabolism is known to have an altered phenotype whereby they primarily respire through lactate production in a process known as the ‘Warburg Effect’ [25–27]. This alteration in metabolism is a key hallmark of cancer cell progression and has been linked to an alteration in mitochondria function [9, 25]. W. somnifera has been reported to induce mitochondrial dysfunction in human leukaemia cells and also reduce mitochondrial function in breast cancer cells [1, 19]. Investigating this mitochondrial alteration further could highlight the value of W. somnifera as an anti-tumour agent. Mitochondrial dysfunction can alter ROS levels, ATP production and overall cell viability and is a novel key target in cancer treatment [9, 13, 14]. In this study we investigated the potential of W. somnifera as a ‘priming’ agent, and showed that ‘priming’ with this root extract enhanced the efficacy of cisplatin through increased ROS in cancer cells while having no detectable effect on non-cancer cells.
Materials and Methods Extraction The extraction method was performed according to the British Pharmacopeia. Withania somnifera root powder (1.0 g, Lot no. 6051SS/03, Pukka, UK), was shaken with 2 mL of dilute ammonia R4. Methanol (20 mL) was added and the mixture was sonicated for 20 minutes. It was then heated on the water bath for 3 minutes and filtered. The filtrate was evaporated to dryness at 60˚C. A stock solution of 0.08335 g of dry extract /mL DMSO was prepared for biological studies.
HPTLC The dry extract was reconstituted in methanol and filtered. The methanolic extract was then applied to a precoated silica gel 60 F254 high performance plate (Merck). CAMAG HPTLC System (Automatic TLC Sampler 4; ADC2 Automatic Developing Chamber; TLC Visualizer; Chromatogram Immersion Device III; TLC Plate Heater III; VisionCats software) was used. Application: 2 μL of reference and test solutions. Mobile phase: Toluene, ethyl acetate, formic acid 10:3:1 (v/v/v). Derivatization: 5% sulphuric acid methanol. Dip (time 0, speed 5), heat at 110˚C for 2 min, detection at UV 366 nm.
HPLC The methanolic root extract (0.3 mg/mL) was analysed using Waters ACQUTTY UPLC— Synapt G2 QTOF (Waters, USA) and a BEH C18 (2.1×100 mm, 1.7 μm) column (Waters, USA) at 40˚C to provide efficiency to the peaks. The mobile phase consisted of water (A) and methanol (B), which were applied in the following gradient elution: from 60:40 (A:B) in 15 min to 20:80(A:B). The flow rate and sample volume were set to 0.3 mL/min. The sample volume was 10 μL. The ESI source was operated in positive (ESI+) ionization mode. The optimized conditions to trigger maximum response of metabolites were listed as follows: capillary voltage, +3 kV; sample cone, + 30 V; extraction cone, +4.0 V; source temperature, 120˚C;
PLOS ONE | DOI:10.1371/journal.pone.0170917 January 27, 2017
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’Priming’ with Withania somnifera
desolvation temperature, 350˚C; cone gas (nitrogen) flow, 50 L/h; and desolvation gas (nitrogen) flow, 600 L/h. Argon was used as collision gas. Leucine- enkephalin (2 ng/mL) was used as the lock mass generating a reference ion at m/z of 556.2771 by a lockspray at 5 μL/min to acquire accurate mass during analysis.
Cell culture Human breast cancer cell line MDA-MB231 and HT-29 colon cancer cells were grown in DMEM (Sigma, UK) with 10% FBS (Sigma), 2% L-Glutamine (Invitrogen, UK) and 2% Penicillin/Streptomycin (P/S) solution (Invitrogen). MDA-MB231 cells were obtained from Dr. T. Kalber (Medical Research Council Clinical Sciences Centre, London) and HT-29 cells were donated by Dr. N. Haiji (Imperial College London). Human non-cancer breast epithelial MCF10A cells were grown in DMEM:F12 (Life Sciences, UK) supplemented with 5% horse serum (Sigma), 2% P/S, 20 ng/mL epidermal growth factor (Sigma), 0.5 mg/mL hydrocortisone (Sigma), 100 ng/mL cholera toxin (Sigma), 10 μg/mL insulin (Sigma). MCF10A cells were donated by Dr. N. Haiji (Imperial College London). All cells were maintained at 37˚C in a humidified 5% CO2 atmosphere.
Treatment conditions All cells were treated with W. somnifera extract (0 μg/mL -10 μg/mL) for 48 h prior to assessment. Primed cells were treated with W. somnifera extract (0 μg/mL -10 μg/mL) for 48 h, washed with PBS and incubated with 100 μM Cisplatin for a further 24 h. ‘Priming’ with quercetin was carried out as follows with 24 h of 40 μM quercetin followed by 100 μM cisplatin for 24 h [11, 28].
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay Cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, UK). All cells were seeded in a 96-well plate at a density between 1.5–3 x104 cells per well and were treated with the various treatment conditions as described above. Cell viability was assayed according to the manufacturers’ protocol. Absorbance was determined at 570 nm and normalised with 690 nm background with a micro plate reader (Spectramax 340PC) after 3.25 h. The optical density was measured as a percentage of the control.
ROS assay Cellular ROS was measured using a 2’,7’–dichlorofluorescein diacetate (DCFDA) Assay (Abcam, UK). The fluorescence was detected on a fluorescent plate reader (FLUOstar Omega, BMG Labtech, UK) with an excitation of 495 nm and emission of 529 nm. Cells were seeded at 2 x104 in 96-well black bottom plate. DCFDA incubation was carried out according to the protocol. Cells were treated using the various treatment conditions previously described. The optical density was measured as a percentage of the control and normalised to cell count.
ATP production, proton leak and mitochondrial respiration ATP, proton leak and mitochondrial/non-mitochondrial respiration was measured using a Seahorse Bioanalyser (Seahorse Biosciences, USA). MDA-MB231, HT-29 and MCF10A cells were seeded 1–2.5 x104 cells per well in a specific Seahorse Bioanalyser 24 well plate and treated appropriately as previously described. The protocol was carried out as specified by the
PLOS ONE | DOI:10.1371/journal.pone.0170917 January 27, 2017
3 / 13
’Priming’ with Withania somnifera
manufacturer’s instructions. Oligomycin (1 μg), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (0.2 μM: MDA-MB231, 0.4 μM: MCF10A, 0.6 μM: HT-29) and antimycin/rotenone (0.25 μM) were added to the sensor plate in the appropriate dilutions directly prior to the commencement of the calibration and assay. Calculations were normalised to protein level using a standard Bradford assay (Biorad).
Assessment of drug-drug interactions To assess the effects of the combination of W. somnifera and cisplatin synergy or antagonism was calculated according to the methods described by Prichard and Shipman [29]. The raw data of 5 experiments was analysed at the 95% confidence interval using the MacSynergy II software developed by Prichard and Shipman. The program calculates the synergy or antagonism of the drug interactions by calculating theoretical additive interactions from the doseresponse surface.
Statistical analysis Statistical analysis was carried out using a one-way ANOVA with Tukey correction and one sample t-test on Graphpad, Prism Software. Results are presented as mean ± SEM. Significance taken when p
Withania somnifera Root Extract Enhances Chemotherapy through ‘Priming’ Aine Brigette Henley1¤, Ling Yang2, Kun-Lin Chuang2, Meliz Sahuri-Arisoylu1, LiHong Wu3, S. W. Annie Bligh1*, Jimmy David Bell1 1 Department of Life Sciences, Faculty of Science and Technology, University of Westminster, London, United Kingdom, 2 Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung, Taiwan, 3 Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China ¤ Current address: Department of Microbiology, Tumour and Cell Biology, Karolinska Institutet, Stockholm, Sweden * [email protected]
a1111111111 a1111111111 a1111111111 a1111111111 a1111111111
OPEN ACCESS Citation: Henley AB, Yang L, Chuang K-L, SahuriArisoylu M, Wu L-H, Bligh SWA, et al. (2017) Withania somnifera Root Extract Enhances Chemotherapy through ‘Priming’. PLoS ONE 12(1): e0170917. doi:10.1371/journal.pone.0170917
Abstract Withania somnifera extracts are known for their anti-cancerous, anti-inflammatory and antioxidative properties. One of their mechanisms of actions is to modulate mitochondrial function through increasing oxidative stress. Recently ‘priming’ has been suggested as a potential mechanism for enhancing cancer cell death. In this study we demonstrate that ‘priming’, in HT-29 colon cells, with W. somnifera root extract increased the potency of the chemotherapeutic agent cisplatin. We have also showed the W. somnifera root extract enhanced mitochondrial dysfunction and that the underlying mechanism of ‘priming’ was selectively through increased ROS. Moreover, we showed that this effect was not seen in non-cancerous cells.
Editor: Irina V Lebedeva, Columbia University, UNITED STATES Received: August 22, 2016
Introduction
Accepted: January 12, 2017
Cancer is one of the major causes of death around the globe, despite the progress observed in surgery, radiation and chemotherapy [1–3]. Cancer progression relies on the ability of cancer cells to exploit the normal physiological processes of the host [1]. This unfortunately means that the cytotoxicity of chemotherapeutic drugs is not limited to cancer cells and can affect non-cancer cells [1, 4]. Today the most common therapeutic strategy of chemotherapy drugs is the use of two drugs in combination, however this is invariably associated with side effects which include chemoresistance [5–7]. It is therefore essential to explore and enhance current methods of chemotherapy to improve their efficacy while also reducing side effects. One such approach is ‘priming’, whereby cancer cells are pre-treated with a ‘priming’ agent (curcumin, quercetin, aspirin) prior to chemotherapy treatment [8–12]. The underlying mechanism underpinning ‘priming’ appears to be the enhancement of cell death through mitochondrial dysfunction [8, 9]. Mitochondrial dysfunction can alter ROS levels, ATP production and overall cell viability and is a novel key target in cancer treatment [9, 13, 14]. Withania somnifera is an Ayurvedic medicinal plant whose root and leaves extracts have been used for its antioxidant and restorative properties as well as to reduce cancer growth
Published: January 27, 2017 Copyright: © 2017 Henley et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors received no specific funding for this work. Competing Interests: The authors have declared that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0170917 January 27, 2017
1 / 13
’Priming’ with Withania somnifera
[2, 6, 15]. W. somnifera extracts have been found to be effective in treating several types of cancer including skin, leukaemia, breast, colon and pancreas [15–21]. However, the mechanisms of action have yet to be fully elucidated, but indications of involvement in mitochondrial membrane permeability have been reported in several studies [1, 21–23]. Additionally, W. somnifera extracts have been shown to increase reactive oxygen species (ROS) [1, 19, 23]. The mitochondria is an important regulator of cell survival and progression and is the main source of ROS which is linked with mitochondrial function [24]. Cancer cells metabolism is known to have an altered phenotype whereby they primarily respire through lactate production in a process known as the ‘Warburg Effect’ [25–27]. This alteration in metabolism is a key hallmark of cancer cell progression and has been linked to an alteration in mitochondria function [9, 25]. W. somnifera has been reported to induce mitochondrial dysfunction in human leukaemia cells and also reduce mitochondrial function in breast cancer cells [1, 19]. Investigating this mitochondrial alteration further could highlight the value of W. somnifera as an anti-tumour agent. Mitochondrial dysfunction can alter ROS levels, ATP production and overall cell viability and is a novel key target in cancer treatment [9, 13, 14]. In this study we investigated the potential of W. somnifera as a ‘priming’ agent, and showed that ‘priming’ with this root extract enhanced the efficacy of cisplatin through increased ROS in cancer cells while having no detectable effect on non-cancer cells.
Materials and Methods Extraction The extraction method was performed according to the British Pharmacopeia. Withania somnifera root powder (1.0 g, Lot no. 6051SS/03, Pukka, UK), was shaken with 2 mL of dilute ammonia R4. Methanol (20 mL) was added and the mixture was sonicated for 20 minutes. It was then heated on the water bath for 3 minutes and filtered. The filtrate was evaporated to dryness at 60˚C. A stock solution of 0.08335 g of dry extract /mL DMSO was prepared for biological studies.
HPTLC The dry extract was reconstituted in methanol and filtered. The methanolic extract was then applied to a precoated silica gel 60 F254 high performance plate (Merck). CAMAG HPTLC System (Automatic TLC Sampler 4; ADC2 Automatic Developing Chamber; TLC Visualizer; Chromatogram Immersion Device III; TLC Plate Heater III; VisionCats software) was used. Application: 2 μL of reference and test solutions. Mobile phase: Toluene, ethyl acetate, formic acid 10:3:1 (v/v/v). Derivatization: 5% sulphuric acid methanol. Dip (time 0, speed 5), heat at 110˚C for 2 min, detection at UV 366 nm.
HPLC The methanolic root extract (0.3 mg/mL) was analysed using Waters ACQUTTY UPLC— Synapt G2 QTOF (Waters, USA) and a BEH C18 (2.1×100 mm, 1.7 μm) column (Waters, USA) at 40˚C to provide efficiency to the peaks. The mobile phase consisted of water (A) and methanol (B), which were applied in the following gradient elution: from 60:40 (A:B) in 15 min to 20:80(A:B). The flow rate and sample volume were set to 0.3 mL/min. The sample volume was 10 μL. The ESI source was operated in positive (ESI+) ionization mode. The optimized conditions to trigger maximum response of metabolites were listed as follows: capillary voltage, +3 kV; sample cone, + 30 V; extraction cone, +4.0 V; source temperature, 120˚C;
PLOS ONE | DOI:10.1371/journal.pone.0170917 January 27, 2017
2 / 13
’Priming’ with Withania somnifera
desolvation temperature, 350˚C; cone gas (nitrogen) flow, 50 L/h; and desolvation gas (nitrogen) flow, 600 L/h. Argon was used as collision gas. Leucine- enkephalin (2 ng/mL) was used as the lock mass generating a reference ion at m/z of 556.2771 by a lockspray at 5 μL/min to acquire accurate mass during analysis.
Cell culture Human breast cancer cell line MDA-MB231 and HT-29 colon cancer cells were grown in DMEM (Sigma, UK) with 10% FBS (Sigma), 2% L-Glutamine (Invitrogen, UK) and 2% Penicillin/Streptomycin (P/S) solution (Invitrogen). MDA-MB231 cells were obtained from Dr. T. Kalber (Medical Research Council Clinical Sciences Centre, London) and HT-29 cells were donated by Dr. N. Haiji (Imperial College London). Human non-cancer breast epithelial MCF10A cells were grown in DMEM:F12 (Life Sciences, UK) supplemented with 5% horse serum (Sigma), 2% P/S, 20 ng/mL epidermal growth factor (Sigma), 0.5 mg/mL hydrocortisone (Sigma), 100 ng/mL cholera toxin (Sigma), 10 μg/mL insulin (Sigma). MCF10A cells were donated by Dr. N. Haiji (Imperial College London). All cells were maintained at 37˚C in a humidified 5% CO2 atmosphere.
Treatment conditions All cells were treated with W. somnifera extract (0 μg/mL -10 μg/mL) for 48 h prior to assessment. Primed cells were treated with W. somnifera extract (0 μg/mL -10 μg/mL) for 48 h, washed with PBS and incubated with 100 μM Cisplatin for a further 24 h. ‘Priming’ with quercetin was carried out as follows with 24 h of 40 μM quercetin followed by 100 μM cisplatin for 24 h [11, 28].
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay Cell viability was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, UK). All cells were seeded in a 96-well plate at a density between 1.5–3 x104 cells per well and were treated with the various treatment conditions as described above. Cell viability was assayed according to the manufacturers’ protocol. Absorbance was determined at 570 nm and normalised with 690 nm background with a micro plate reader (Spectramax 340PC) after 3.25 h. The optical density was measured as a percentage of the control.
ROS assay Cellular ROS was measured using a 2’,7’–dichlorofluorescein diacetate (DCFDA) Assay (Abcam, UK). The fluorescence was detected on a fluorescent plate reader (FLUOstar Omega, BMG Labtech, UK) with an excitation of 495 nm and emission of 529 nm. Cells were seeded at 2 x104 in 96-well black bottom plate. DCFDA incubation was carried out according to the protocol. Cells were treated using the various treatment conditions previously described. The optical density was measured as a percentage of the control and normalised to cell count.
ATP production, proton leak and mitochondrial respiration ATP, proton leak and mitochondrial/non-mitochondrial respiration was measured using a Seahorse Bioanalyser (Seahorse Biosciences, USA). MDA-MB231, HT-29 and MCF10A cells were seeded 1–2.5 x104 cells per well in a specific Seahorse Bioanalyser 24 well plate and treated appropriately as previously described. The protocol was carried out as specified by the
PLOS ONE | DOI:10.1371/journal.pone.0170917 January 27, 2017
3 / 13
’Priming’ with Withania somnifera
manufacturer’s instructions. Oligomycin (1 μg), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (0.2 μM: MDA-MB231, 0.4 μM: MCF10A, 0.6 μM: HT-29) and antimycin/rotenone (0.25 μM) were added to the sensor plate in the appropriate dilutions directly prior to the commencement of the calibration and assay. Calculations were normalised to protein level using a standard Bradford assay (Biorad).
Assessment of drug-drug interactions To assess the effects of the combination of W. somnifera and cisplatin synergy or antagonism was calculated according to the methods described by Prichard and Shipman [29]. The raw data of 5 experiments was analysed at the 95% confidence interval using the MacSynergy II software developed by Prichard and Shipman. The program calculates the synergy or antagonism of the drug interactions by calculating theoretical additive interactions from the doseresponse surface.
Statistical analysis Statistical analysis was carried out using a one-way ANOVA with Tukey correction and one sample t-test on Graphpad, Prism Software. Results are presented as mean ± SEM. Significance taken when p
